Neurons affected by neurodegenerative disease exhibit dysfunction of both mitochondria and the ubiquitin- proteasome system, suggesting a functional link between the two. Understanding the interplay between these two systems is crucial for comprehending the molecular mechanisms of disease progression. However, specific mitochondrial E3 ligases and their substrates have yet to be identified, despite evidence that mitochondria contain all the components necessary for ubiquitination of mitochondrial proteins. Additionally, little is known about the function of ubiquitination in mitochondria, let alone how ubiquitination of mitochondrial proteins plays a role in neurodegeneration. In her preliminary studies the candidate has identified an E3 ligase, RING finger protein 11 (RNF11), which translocates to mitochondria and interacts with a mitochondrial protein. RNF11 is a ubiquitously expressed protein originally identified in a search for novel proteins containing a RING finger, a motif that has been implicated as a key determinant of E3 ligase activity. The candidate hypothesizes that RNF11 is a mitochondrial E3 ligase that plays a role in cell survival. This hypothesis will be thoroughly tested by first characterizing the association of RNF11 with the identified mitochondrial interactor, a component of complex III, as well as RNF11-mediated ubiquitination of that interactor (Aim 1). The functional consequences of this interaction will be assessed by quantifying the role of RNF11 in modulating complex III function and in suppression of mitochondria-dependent apoptosis (Aim 2). The interaction of endogenous RNF11 and the complex III protein will be confirmed using co-immunoprecipitation and in vitro binding. RNF11-mediated ubiquitination of the interactor will be examined by altering levels of RNF11 protein by overexpression and knockdown via RNAi and examining the levels of ubiquitination of the immunoprecipitated complex III protein. Overexpression and knockdown will also be used to examine the effect of RNF11 protein levels on the activity of complex III and on the initiation of apoptosis in in vitro models of neurodegenerative disease. The molecular processes that lead to cell death in Parkinson's disease are not well understood. This proposal aims to describe how one protein, RNF11, plays a role in cell death. Understanding the cellular events that affect cell death will enable future researchers to identify molecular targets for treatment of the disease. [unreadable] [unreadable] [unreadable]